Effect of milrinone analogues on intracellular calcium increase in single living H9C2 cardiac cells

Article abstract of DOI:10.1016/j.ejmech.2010.08.001

The synthesis of milrinone analogues where the 4-pyridyl moiety was replaced by an ester or amide group is reported. Only amide derivatives are able to support intracellular calcium influx following chemical depolarization with 60 mM KCl in a percentage varying from 20 to 45% of differentiated H9C2 cardiomyocytes. Those cells were differentiated after chronic exposure to 10 nM retinoic acid which induces the expression of voltage-gated calcium channels. Analogues of milrinone containing an ester function did not show significant activity.

Full text of DOI:10.1016/j.ejmech.2010.08.001

European Journal of Medicinal Chemistry 45 (2010) 4928e4933
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Effect of milrinone analogues on intracellular calcium increase in single living
H9C2 cardiac cells
,
c
,
,
,
,
,c
Tiziana Pietrangelo b 1, Letizia Giampietro a 1, Barbara De Filippis a 1, Rita La Rovere b c, Stefania Fulle b
,
,
Rosa Amoroso a
*
a Dipartimento di Scienze del Farmaco, Università degli Studi “G. d’Annunzio”, via dei Vestini 31, 66100 Chieti, Italy
b Dipartimento di Scienze Mediche di Base e Applicate, Università degli Studi “G. d’Annunzio”, via dei Vestini 31, 66100 Chieti, Italy
c Interuniversity Institute of Myology (IIM), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of milrinone analogues where the 4-pyridyl moiety was replaced by an ester or amide
group is reported. Only amide derivatives are able to support intracellular calcium influx following
chemical depolarization with 60 mM KCl in a percentage varying from 20 to 45% of differentiated H9C2
cardiomyocytes. Those cells were differentiated after chronic exposure to 10 nM retinoic acid which
induces the expression of voltage-gated calcium channels. Analogues of milrinone containing an ester
function did not show significant activity.
Received 13 March 2010
Received in revised form
27 July 2010
Accepted 1 August 2010
Available online 12 August 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Intracellular Ca2þ concentration
H9C2 cells
Epifluorescent microscopy on living
single cells
Milrinone
Milrinone analogues
1. Introduction
resulting in hemodynamic disturbances and a cascade of local and
systemic changes [4,5]. The [Ca2þ]i homeostasis is clearly abnormal
The role of Ca2þ signaling in cardiac contractile regulation has
been the subject of several reviews [1e3]; in normal car-
diomyocytes, the increase in intracellular Ca2þ concentration
([Ca2þ]i) known as a Ca2þ transient, is the chemical signal that
transduces electrical depolarization to mechanical movement,
a sequence of events collectively known as excitationecontraction
(EC) coupling. The EC coupling is initiated by influx of Ca2þ through
L-type Ca2þ channels (LTCCs) that, in turn, trigger the release of
Ca2þ from the sarcoplasmic reticulum by activation of ryanodine
receptors; Ca2þ diffuses through the cytosolic space to contractile
proteins, and binds to the myofilament protein troponin C, which
activates cardiac contraction. The whole process of Ca2þ movement
is characterized by a transient increase in [Ca2þ]i from 100 nmol/L
in the cardiomyocytes of the failing heart, inducing profound alter-
ations in the ability of the cardiac cells to contract and relax [6]. The
principle objective in treating CHF is to quickly correct hemody-
namic disorders (low cardiac output, poor tissue perfusion of vital
organs, pulmonary congestion) by improving cardiac contractility
with cardiotonic agents; these agents induce a positive inotropic
effect (PIE) by increase of calcium cycling, acting through various
subcellular mechanism, such as b-adrenergic agonism and phos-
phodiesterase 3 (PDE3) inhibition, both of which result in increased
levels of cyclic AMP and [Ca2þ]i in cardiac myocytes [7e9].
In the last decade, it was observed that the [Ca2þ]i transients
inducing the contraction rise more slowly in failing heart, which
may limit synchronous contractile activation [10]; this effect was
attributed to abnormalities in the density and regulation of LTCCs,
that are contributors to the Ca2þ homeostasis in myocytes. Altered
Ca2þ cycling usually precedes the depression of mechanical
performance in CHF, and consequently correction of the disorders
of Ca2þ cycling has potential as a new and intriguing therapeutic
strategy against CHF.
to approximately 1 mmol/L.
Chronic heart failure (CHF) is a condition in which the heart
cannot pump enough blood through the body; it is a clinical
syndrome characterized by a reduced pump efficiency of the heart
* Corresponding author. Tel.: þ39 0871 3554686; fax: þ39 0871 3554911.
E-mail address: ramoroso@unich.it (R. Amoroso).
Milrinone is a potent positive inotropic and vasodilating agent,
and its effects are mainly attributable to PDE3 inhibition, leading to
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.001

T. Pietrangelo et al. / European Journal of Medicinal Chemistry 45 (2010) 4928e4933
4929
N
4
HOOC
CN
O
CN
O
3
CN
O
5
2
N
H
N
H
N
H
6
1
2
1
milrinone
14
R =
R = Et
R =
3
4
O
CN
O
O
15 R =
RO
CN
O
RHN
N
H
Et
N
N
S
N
H
R =
11
R =
R =
16
17
N
12 R =
S
EtOOC
S
Fig. 1. Milrinone and structure of new analogues.
intracellular cyclic AMP accumulation and stimulation of trans-
sarcolemmal calcium influx via the slow calcium channels; it is not
We also decided to replace the ester groups by amides, which
should be better resistant to hydrolytic cleavage during the bio-
logical assay, that involves whole cells in incubation conditions.
Thus, based on inotropic activity of the benzyl ester analogue of
milrinone [16], we decided to explore the biological activity of
a small set of benzylic amides. Finally, a close evaluation of an ethyl
carbamate derivative was also made. As with the amides, this
compound is chemically resistant to hydrolysis.
Preliminary indications about structureeactivity relationships
are discussed, in order to obtain useful information for the design of
new analogues with better pharmacological profiles. Also, cyto-
toxicity was measured using a colorimetric assay, with the aim of
evaluating the potential toxicity of the more active compounds.
a
b-adrenergic agonist nor an inhibitor of sodiumepotassium
adenosine triphosphatase as are the digitalis glycosides [11,12].
Various chemical modifications of milrinone have been carried out,
in order to develop new inotropic agents with improved cardiotonic
activity and to minimize side effects [13e19]; moreover, the publi-
cation of crystal structure of PDE3B [20] revealed important infor-
mation about the molecular features required for the design of more
potent and selective inhibitors [21]. Analogues of milrinone, in
which various chemical modifications were carried out in positions
2, 3, and 5, were synthesized by the Mosti group, and provided
important information about structureeactivity relationships (SAR)
[13,16]; in these studies, replacing the 4-pyridyl group by an ester
increased PDE3 antagonism by 2e4 fold. This interesting result
prompted us to investigate new structural modifications at the 3-
position of milrinone, where the 4-pyridyl moiety was simply
removed or replaced with an acid, ester or amide group (Fig. 1). We
tested new compounds for their effects on [Ca2þ]i in H9C2 cardiac
cells, by using the fluorescent microscopy on living single cell. The
analogue 1, notsubstituted in the 3-position, the acid 2, and the ethyl
and benzyl esters 3 and 4, respectively, are known in the literature
and they were previously tested as cardiotonic agents [13,16];
however, we have included them as they have not previously been
subjected to our test system. The ester 11, containing a pyridin-2-
ylthioethyl chain, was designed taking into account a docking study
on a series of PDE3 inhibitors [21,22], with the aim of exploring the
influenceofa spacer betweenthe pyridoneand theheterocyclic ring,
2. Results and discussion
2.1. Chemistry
Compound 1 was tested as received by SigmaeAldrich, while
acid 2 and esters 3 and 4 were synthesized according to the liter-
ature procedures [13,16]. Compounds 11 and 12 were prepared as
outlined in Scheme 1. The starting
b-ketoesters 7 and 8 were
obtained by refluxing the ethyl 3-oxobutanoate and the appro-
priate alcohol (5e6) in toluene under Dean Stark conditions; these
intermediates were treated with a slight excess of N,N-dime-
thylformamide dimethylacetal (DMF.DMA), to give compounds 9
and 10 with good yield. At last, treatment of 9 and 10 with a solu-
tion of sodium cyanoacetamide in anhydrous ethanol afforded the
desired esters 11 and 12, which were purified by recrystallization.
The amides 14e17 were accessed by conversion of the acid 2 to
the desired amide functionality as outlined in Scheme 2. The acid 2,
and the substitution of the pyridine with
a benzothiazole
(compound 12) was made to enhance the steric bulk of the aromatic
system.
O
O
O
CN
O
O
O
c
RO
b
a
R
OH
5-6
RO
RO
N
H
N
7-8
11-12
9-10
N
S
5, 7, 9 and 11
6, 8, 10 and 12
R =
N
R =
S
S
Scheme 1. Reagents and conditions: (a) ethyl 3-oxobutanoate, toluene, reflux, 24e48 h; (b) DMF.DMA, DMF, reflux, 5 h; (c) cyanoacetamide, EtONa, rt, 18e24 h.

4930
T. Pietrangelo et al. / European Journal of Medicinal Chemistry 45 (2010) 4928e4933
O
O
O
CN
O
CN
O
CN
O
HO
b
RHN
Cl
a
N
H
N
H
N
H
2
13
14-17
Scheme 2. Reagents and conditions: (a) SOCl2, reflux, 4 h; (b) RNH2, NEt3, AcOEt, CH2Cl2, rt, 6e24 h.
prepared according to the previous method [13], was converted
into the corresponding acid chloride 13 by treatment with SOCl2.
The reaction of 13 with the suitable amine, in the presence of
triethylamine, afforded the amides 14e17, which were purified by
column chromatography on silica gel or recrystallization from
ethanol [23].
little disruption of the cellular functions [27]. The [Ca2þ]i variations
induced by extracellular compounds were indirectly measured by
selecting the ratio intensity fluorescence of emission in the whole
cellular area of different stimulated cells.
We did two different set of experiments on differentiated cells:
the first set was to test if the cells were able to increase the cyto-
plasmic [Ca2þ]i when directly stimulated. We tested a minimum of
10 and a maximum of 25 cells with 10 mM of each molecule and
2.2. Calcium imaging
milrinone; only pyridone 1 was able to induce a basal [Ca2þ
]
i
increase on 78% of stimulated cells.
In this work, we wanted to investigate the ability of new
molecules and milrinone to interfere with the [Ca2þ]i level during
the plasma membrane depolarization in H9C2 cells, an embryonal
cardiomyocytes cellular line from rat ventricle, which induces
specific expression of alpha 1C subtype of L-type calcium channel,
typical of cardiomyocytes; H9C2 cells are able to differentiate in
vitro after chronic exposure to 10 nM retinoic acid [24]. The
chemical depolarization with KCl induced the opening of the LTCCs
and the influx of Ca2þ into the cells. Calcium is a key signaling ion in
excitationecontraction coupling of the cardiac muscle cells and
a good indicator of physiologically relevant activity of new mole-
cules interfering at this level. The measurement of [Ca2þ]i was
made by epifluorescent microscopy on living single cells, a very
useful video-imaging technique that allows the investigation of the
[Ca2þ]i transients in living cells [25]; the result of the experiment is
a series of fluorescence images where the intensities represent the
[Ca2þ]i variation in a living cells of interest [26]. Video-imaging
experiments were performed by incubating cells with fluorescent
indicator Fura 2-AM (Fura 2 pentaacetoxymethyl ester), which forms
a complex with Ca2þ (Kd 224 nM) and has been used commonly to
measure the [Ca2þ]i because it can be loaded into living cells with
Then, in the second set of experiments we tested both doses of 1
and 10 M of each compound, to study the effect on KCl-dependent
[Ca2þ]i variation of the new milrinone derivatives, but we preferred
the dose of 10 M due to the reproducibility of the data. Thus, we
administered 10 M of each compound and milrinone on cells
m
m
m
depolarized by 60 mM KCl application, in the presence of extra-
cellular Ca2þ; in the absence of activators, this depolarization is able
to induce a [Ca2þ]i transient in myocytes, characterized by a rapidly
ascending phase and a return to baseline within 1 min (Fig. 2). The
treatment of 1, 14, 16, and 17 within 30 s after the beginning of the
KCl-dependent plateau phase, was able to significantly extend
the [Ca2þ]i increase; this behavior is similar to milrinone, whose
effect on calcium influx across the plasma membrane is already
known [12]. The others milrinone analogues were unable to induce
these effects.
In order to quantify the above graphical results, in Table 1 we
summarised the percentage of cells responsive to new compounds,
compared with milrinone. We confirmed a positive inotropic effect
of milrinone (50% of responsive cells), already observed by Olson
et al. [12]; a similar result was obtained with compound 1, in which
Fig. 2. The intracellular [Ca2þ
] transients recorded on H9C2 myocytes following the stimulus with 60 mM KCl administered in NES. Each molecule was administered at 10 mM
i
concentration.

T. Pietrangelo et al. / European Journal of Medicinal Chemistry 45 (2010) 4928e4933
4931
activity; however, the amide work is interesting and suggests that
further studies will be needed to rationalise the structureeactivity
relationships (SAR). Synthesis of single enantiomers will also be
very useful in determining what is going on here. Compound 17,
containing an amide and a carbamate group, both resistant to
hydrolysis, affected the calcium transient in a small percentage of
cells (25%); this close evaluation suggests the possibility of further
milrinone modifications, with the aim of obtaining new structures
capable of modifying the calcium influx across the plasma
membrane.
From our studies, we observed that only amide derivatives of
milrinone are able to prolong the KCl-dependent plateau, while
esters are inactive, suggesting that the activity on calcium channels
was strongly influenced from substituent in 4-position of pyr-
idinone ring. Further studies are in progress to clarify the struc-
tureeactivity requirements for such agents.
Fig. 3. The dose dependent cell proliferation. The concentrations were of 0.01, 1, 10 and
100 M.
m
2.3. Toxicity profile
the 4-pyridyl moiety of milrinone was simply removed: calcium
influx was sustained in 45% of investigated cells. On the other case,
the replacement of the 4-pyridyl group with an acid function
(compound 2) did not induce [Ca2þ]i transients in differentiated
cells; this behavior is in accordance with previous literature results,
in which the same compound was scarcely active as an inotropic
agent [13]. Also the esters 3 and 4 were found to be inactive; this
appears to disagree with studies reported in the Introduction
section, that provided evidence of inotropic properties of methyl
and benzyl esters derivatives of milrinone. We postulate that this
result is due to the presence of esterase enzymes in our cell line, as
demonstrated by hydrolytic degradation of fluorescent indicator
Fura 2-AM into Fura 2, which is trapped within cells and is used to
measure intracellular free Ca2þ concentration by a fluorescence
ratio method [27]. Therefore, the esters were probably cleaved by
esterases in our experimental conditions, affording the inactive
acid. We propose the same explanation for more hindered esters 11
and 12; so, we consider deleterious and useless the substitution of
the 4-pyridyl moiety of milrinone with an ester function, at least in
our experimental conditions.
The replacement of the ester groups with amides, which are
likely to be significantly more resistant to hydrolytic cleavage
under the conditions of biological assay, showed an increase in
percentage of responsive cells depending from substitution on
benzylic carbon: among compounds 14e16, only 14 and 16, with
N-benzyl and N-1-phenylpropyl groups, respectively, showed
a good degree of calcium channel activation (40% and 30% of
responsive cells), whereas the N-1-phenylethyl derivative (15) was
found inactive. These data, although very interesting, do not allow
us to rationalise the influence of steric properties on biological
In order to evaluate the potential toxicity of compounds 1, 14, 16,
17, and milrinone, cytotoxicity was measured using the 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
assay [28]; the principle behind this technique depends on the
capacity of living cells to reduce tetrazolium salt to a formazan
crystal in their metabolizing mitochondria. This purple-colored
formazan crystal can be measured using an ELISA reader at
a wavelength of 570 nm and the value of the optical density is
related to the number of viable cells in each sample. The MTT
method is reported to have several advantages with respect to the
speed of analysis, quantification and the management of many
samples. The in vitro test is an informative model of the reactions
that may occur in vivo.
We analysed the cellular vitality of the H9C2 as undifferentiated
or differentiated phenotype in the presence for 24 h of the doses
10 nM, 1, 10 and 100
undifferentiated cells, only the greatest concentration of 100
proved to be toxic; in fact, for compounds 1 and 16 the cell viability
was reduced to 50% of the control, while molecules 14, 17, and
milrinone significantly inhibited the proliferation of H9C2 cells
reducing cell vitality to 20e30% (data not shown).
mM for each molecule and milrinone. On
mM
As showed in Fig. 3, the addition of 10 nM and 1 mM of tested
compounds and milrinone to the differentiated H9C2 cells induced
a light decrease in MTT activity, with cell viability percentage
around 90%. At concentrations of 10
a slight cytotoxic effect with percentage of inhibition from 75 to
84%. It is worth mentioning that 10 M is slightly toxic after 24 h of
incubation, but this effect was not been observed during the fast
depolarization used for video-imaging experiments. Finally,
mM, all compounds showed
m
concentrations of 100
mM were found to be toxic, in fact they
reduced the cell viability below 50%. The toxicity could be due to
different causes that at this stage of our study we ignore; we can
suppose that the toxicity did not strictly depend on the differen-
tiation state because the response of undifferentiated and differ-
entiated cells, except for the highest dose, was quite similar. The
data deriving from cytotoxicity test will be really important in the
development of new inotropic compounds and we will take into
account of them for future investigations.
Table 1
Percentage of differentiated H9C2 that prolonged the KCl-dependent [Ca2þ]i tran-
sient in the presence of new compounds and milrinone.
Compounda
% Response
nb
1
2
3
4
11
12
14
15
45
0
0
0
0
0
40
0
30
25
50
18
15
10
10
14
11
25
10
18
22
17
3. Conclusions
New milrinone analogues were synthesized by removing the
pyridyl moiety or replacing it with an ester or amide function and
their ability to interfere with the [Ca2þ]i level during the plasma
membrane depolarization in H9C2 cardiomyocytes was tested. The
cells were differentiated after chronic exposure to 10 nM retinoic
16
17
Milrinone
a
Concentration of 10 mM.
Number of tested cells.
b

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T. Pietrangelo et al. / European Journal of Medicinal Chemistry 45 (2010) 4928e4933
acid which induces the expression of voltage-gated calcium chan-
nels. Among newly synthesized molecules, only amide derivatives,
of NaOEt [from Na (5.2 mmol) and anhydrous EtOH (16 mL)] to
cyanoacetamide (5.2 mmol) dissolved in warm anhydrous EtOH
(32 mL). The mixture was stirred at room temperature for 18e21 h.
The residue was dissolved in a minimum amount of H2O and
extracted with Et2O (3 ꢁ 35 mL). The aqueous solution was acidified
with 6 M HCl (pH ¼ 1) at 0 ꢂC. The precipitate was filtered, washed
with water, dried in an oven at 100 ꢂC and recrystallized from 95%
EtOH to give 11e12.
incubated at concentration of 10 mM in the presence of the KCl-
dependent [Ca2þ]i transient of cardiac myocytes are able to extend
it in a significant manner. Further studies are in progress to define
the structureeactivity requirements and the mechanisms of action
of the above-mentioned compounds.
4. Experimental protocols
2-(Pyridin-2-ylthio)ethyl 5-cyano-2-methyl-6-oxo-1,6-dihydro-
pyridine-3-carboxylate (11): orange solid, 66% yield, mp 159e161 ꢂC.
4.1. Chemistry
IR (KBr) 3447, 2229, 1718, 1654 cmꢀ1; 1H NMR (DMSO-d6)
d 2.55 (s,
3H, CH3), 3.52 (t, 2H, CH2S), 4.39 (t, 2H, OCH2), 7.1 (t, 1H, CHAr), 7.33
(d, 1H, CHAr), 7.63 (t, 1H, CHAr), 8.24 (s, 1H, ]CH), 8.40 (d,
Melting points were determined on a Büchi B-540 apparatus
and are uncorrected. Infrared spectra were recorded on an FT-IR
1600 PerkineElmer spectrometer. NMR spectra were run at
1H, CHAr); 13C NMR (DMSO-d6)
d 20.20 (CH3), 28.30 (CH2S),
64.31 (OCH2), 100.31 (CCN), 107.61 (CCOO), 116.28 (CCN), 120.81,
122.81, 137.49 and 150.09 (CHAr), 149.43 (]CH), 157.77 (SCAr),
160.39 (HNCO), 160.63 (CCH3), 163.52 (O]CO). Anal. calcd for
C15H13N3O3S: C, 57.13; H, 4.16; N, 13.33. Found: C, 57.06; H, 4.12;
N, 13.34.
300 MHz on a Varian instrument; chemical shifts (d) are reported in
ppm. Microanalyses were carried out with a Eurovector Euro EA
3000 model analyzer and the analytical results are within 0.4% of
the theoretical values. GC analyses were run on an autosystem GC
PerkineElmer apparatus using a fused silica capillary column
(30 m, 0.53 mm ID), SPB-5 Supelco. Commercial reagents were used
as received from Aldrich or Fluka.
2-(1,3-Benzothiazol-2-ylthio)ethyl 5-cyano-2-methyl-6-oxo-1,6-
dihydropyridine-3-carboxylate (12): pink solid, 40% yield, mp 213 ꢂC
dec. IR (KBr) 2218, 3447,1718,1654 cmꢀ1; 1H NMR (DMSO-d6)
d 2.54
5-cyano-2-methyl-6-oxo-1,6-dihydropyridine (1), 5-cyano-2-
methyl-6-oxo-1,6-dihydro-3-pyridinecarboxylic acid (2), ethyl 5-
cyano-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate (3), and
(s, 3H, CH3), 3.75 (t, 2H, CH2S), 4.55 (t, 2H, OCH2), 7.29e7.35 (m, 1H,
CHAr), 7.38e7.43 (m, 1H, CHAr), 7.74e7.77 (d, 1H, CHAr), 7.94e7.97
(m, 1H, CHAr); 13C NMR (DMSO-d6)
d 20.18 (CH3), 32.35 (CH2S),
benzyl
5-cyano-2-methyl-6-oxo-1,6-dihydropyridine-3-carbox-
63.81 (OCH2), 100.56 (CCOO), 107.39 (CCN), 116.15 (CCN), 121.65 and
127.06 (CHAr), 135.27 (CAr), 149.23 (]CH), 153.10 (CAr), 160.35
(HNCO), 160.46 (CCH3), 163.39 (O]CO), 166.55 (SCS). Anal. calcd for
C17H13N3O3S2: C, 54.97; H, 3.53; N, 11.31. Found: C, 54.94; H, 3.55;
N, 11.30.
ylate (4) were synthesized as reported in the literature [13,16].
4.1.1. General procedure for the preparation of b-ketoesters 6e7
In a Dean Stark apparatus, a stirred mixture of the suitable
alcohol (5e6) (1.2 eq) and ethyl 3-oxobutanoate (7.9 mmol) in
toluene (55 mL) was heated until no starting material remained.
After 22 h the reaction mixture was concentrated under reduced
pressure to afford the crude product, which was purified by column
chromatography on silica gel (eluent cyclohexane/EtOAc 95:5) to
afford 7e8.
4.1.4. General procedure for the preparation of acid chloride 13
The acid 2 (200 mg, 1.2 mmol) was dissolved in SOCl2 (57 eq,
63.0 mmol, 4.6 mL) and heated at reflux for 3e4 h. The solvent was
evaporated and the residue was azeotroped with toluene (4 mL) to
give acid chloride 13 as white solid, that was used without further
purification.
2-(Pyridin-2-ylthio)ethyl 3-oxobutanoate (7): yellow oil, 60%
yield. IR (KBr) 1743, 1717 cmꢀ1; 1H NMR (CDCl3)
d
2.27 (s, 3H, CH3),
3.44e3.47 (m, 4H, COCH2CO and CH2S), 4.40 (t, 2H, OCH2),
6.97e8.42 (m, 4H, CHAr); 13C NMR (CDCl3)
28.30 (CH2S), 30.45
4.1.5. General procedure for the preparation of amides 14e17
The acid chloride 13 was dissolved in EtOAc (4 mL) and added
dropwise to a solution of suitable amine (1 eq) and NEt3 (0.5 eq,
0.08 mL) in CH2Cl2 (4 mL). The mixture was stirred at room
temperature for 6e24 h and the solvent was evaporated. The
residue was purified by column chromatography on silica gel
(eluent cyclohexane:EtOAc 3:7 or CH2Cl2:MeOH 9:1) or by crys-
tallization from EtOH to give desired amide 14e17.
d
(CH3), 50.20 (COCH2CO), 64.30 (OCH2), 119.98, 122.63, 136.36 and
149.68 (CHAr), 157.58 (CAr), 167.18 (O]CO), 200.69 (CH3CO). Anal.
calcd for C11H13NO3S: C, 55.21; H, 5.48; N, 5.85. Found: C, 55.01; H,
5.45; N, 5.65.
2-(1,3-Benzothiazol-2-ylthio)ethyl 3-oxobutanoate (8): colourless
oil, 59% yield. IR (KBr) 1743, 1714 cmꢀ1 1H NMR (CDCl3)
; d 2.26 (s,
3H, CH3), 3.47 (s, 2H, COCH2CO), 3.64 (t, 2H, CH2S), 4.54 (t, 2H,
OCH2), 7.28e7.44 (m, 2H, CHAr), 7.74e7.77 (d, 1H, CHAr), 7.84e7.88
N-benzyl-5-cyano-2-methyl-6-oxo-1,6-dihydropyridine-3-car-
boxamide (14): white solid, 41% yield, mp 260 ꢂC dec. IR (KBr) 2237,
(d, 1H, CHAr); 13C NMR (CDCl3)
d
30.45 (CH3), 31.74 (CH2S), 50.07
1678, 1633 cmꢀ1
;
1H NMR (DMSO-d6)
d
2.43 (s, 3H, CH3), 4.36 (d,
2H, CH2), 7.27e7.31 (m, 5H, CHAr), 8.25 (s, 1H, CH), 8.75 (br s, NH);
13C NMR (DMSO-d6)
19.07 (CH3), 43.32 (CH2Ar), 99.66 (CCONH),
(COCH2CO), 63.66 (OCH2), 121.28, 121.84, 124.69 and 126.36 (CHAr),
135.55 and 153.22 (CAr), 165.49 (]CeS), 167.06 (OC]O), 200.45
(CH3CO); Anal. calcd for C13H13NO3S2: C, 52.86; H, 4.44; N, 4.74.
Found: C, 52.75; H, 4.47; N, 4.69.
d
113.68 (CCN), 113.82 (CN), 127.52 (Ar), 128.01 (Ar), 128.99 (Ar),
139.76 (CArCO), 148.56 (HC]C), 155.49 (CONH), 160.57 (CON-
HCH2Ar), 164.99 (CCH3). Anal. calcd for C15H13N3O2: C, 67.40; H,
4.90; N, 15.72. Found: C, 67.36; H, 4.89; N, 15.68.
4.1.2. General procedure for the preparation of 2-
dimethylaminomethylene-1,3-diones 9e10
5-Cyano-2-methyl-6-oxo-N-(1-phenylethyl)-1,6-dihydropyr-
A solution of 7e8 (4.7 mmol) in DMF.DMA (1.2 eq) was refluxed
for 5 h. The excess of acetal was distilled off under reduced pressure
and the crude product (9e10) was used without further purification.
idine-3-carboxamide (15): white solid, 48% yield, mp 240 ꢂC dec.
IR (KBr) 2235, 1680, 1640 cmꢀ1; 1H NMR (DMSO-d6)
d
2.43 (s, 3H,
CH3), 4.36 (d, 2H, CH2), 7.27e7.31 (m, 5H, CHAr), 8.25 (s, 1H, CH),
8.75 (br s, NH); 13C NMR (DMSO-d6)
14.87 (CH2CH3), 18.99 (CH3),
d
4.1.3. General procedure for the preparation of dihydropyridines
11e12
The acrylate 9e10 (4.9 mmol) dissolved in anhydrous EtOH
(16 mL), was added at room temperature to a solution of sodium
cyanoacetamide in anhydrous EtOH, prepared by adding a solution
55.57 (CHCH3), 99.60 (CCONH), 114.00 (CCN), 116.91 (CN), 127.17
(CAr), 127.40 (CAr), 128.92 (CAr), 144.15 (CArCO), 148.55 (HC]C),
155.68 (CONH), 160.60 (CONH), 164.49 (CCH3). Anal. calcd for
C16H15N3O2: C, 68.31; H, 5.37; N, 14.94. Found: C, 68.35; H, 5.32;
N, 11.90.

T. Pietrangelo et al. / European Journal of Medicinal Chemistry 45 (2010) 4928e4933
4933
5-Cyano-2-methyl-6-oxo-N-(1-phenylpropyl)-1,6-dihydropyr-
attached, incubated with new compounds and milrinone at
different concentrations: 10 nM, 1 M, 10 M and 100 M for 24 h.
At the end of the incubation the GM or DM was removed and the
cells were treated with 20
l of MTT solution (5 mg mLꢀ1 in PBS) to
each well, followed by incubation at 37 ꢂC for 3 h. The supernatant
idine-3-carboxamide (16): white solid, 56% yield, mp 250 ꢂC dec. IR
m
m
m
(KBr) 2233, 1673, 1534 cmꢀ1 1H NMR (DMSO-d6)
; d 0.84 (t, 3H,
CH3CH2), 1.71 (m, 2H, CH2CH3), 2.36 (s, 3H, CH3), 4.74 (m, 1H, CH),
m
7.28e7.33 (m, 5H, CHAr), 8.24 (s, 1H, CH), 8.75 (br s, NH); 13C NMR
(DMSO-d6)
d
11.87 (CH2CH3), 18.99 (CH3), 29.80 (CH2CH3), 55.57
was removed, and 200 ml DMSO was added to each well. The plate
(CHCH2CH3), 99.60 (CCONH),114.00 (CCN),116.91 (CN),127.17 (CAr),
127.40 (CAr), 128.92 (CAr), 144.15 (CArCO), 148.55 (HC]C), 155.68
(CONH), 160.60 (CONH), 164.49 (CCH3). Anal. calcd for C17H17N3O2:
C, 69.14; H, 5.80; N, 14.23. Found: C, 69.11; H, 5.77; N, 14.20.
Ethyl 4-{[(5-cyano-2-methyl-6-oxo-1,6-dihydropyridin-3-yl)car-
bonyl]amino}piperidine-1-carboxylate (17): white solid, 42% yield,
mp 190 ꢂC dec. IR (KBr) 2239, 1673, 1534 cmꢀ1; 1H NMR (DMSO-d6)
was agitated for 5 min, and incubated for 30 min at 37 ꢂC. Finally,
the plate was read at 540 nm on a Titertek Multiscan Microelisa
Reader (Flow Laboratories, Urvine, UT, USA) [29].
Acknowledgements
This study was supported by University “G. d’Annunzio” of
Chieti local grants.
d
1.15 (t, 3H, CH2CH3), 1.12e1.37 (m, 2H, CH2), 1.74e1.78 (m, 2H,
CH2), 2.41 (s, 3H, CH3), 2.90 (br NH), 3.29e3.3 (m, 2H, CH2),
3.85e3.89 (m, 2H, CH2), 4.00 (q, 2H, CH2CH3), 8.19 (s, 1H, CH); 13C
NMR (DMSO-d6)
d 15.2 (CH3), 18.92 (CH3), 31.67 (CH2CCH2), 42.94
References
(CH2NCH2), 46.80 (CH), 61.32 (CH2), 99.58 (CCN), 114.04 (CC]O),
116.84 (CN), 148.68 (HC]C), 155.30 (C]O), 155.60 (COO), 160.58
(CON), 164.29 (CH3C]C). Anal. calcd for C16H20N4O4: C, 57.82;
H, 6.07; N, 16.86. Found: C, 57.80; H, 6.01; N, 16.88.
[1] M. Endoh, J. Pharmacol. Sci. 100 (2006) 525e537.
[2] T. Kobayashi, L. Jin, P.P. de Tombe, Pflugers Arch. e Eur. J. Physiol. 457 (2008)
37e46.
[3] D.M. Bers, Annu. Rev. Physiol. 70 (2008) 23e49.
[4] H. Krum, W.T. Abraham, Lancet 373 (2009) 941e955.
[5] M.M. Redfield, N. Engl. J. Med. 437 (2002) 1442e1444.
[6] D.M. Bers, Physiology 21 (2006) 380e387.
4.2. Biology
[7] U. Landmesser, H. Drexler, Curr. Treat. Options Cardiovasc. Med. 9 (2007)
443e449.
[8] H.H. Rasmussen, G.A. Figtree, H. Krum, H. Bundgaard, Curr. Opin. Investig.
Drugs 10 (2009) 955e962.
[9] P.E. Thompson, V. Manganiello, E. Degerman, Curr. Top. Med. Chem. 7 (2007)
421e436.
[10] T.J. Kamp, J.-Q. He, Circ. Res. 91 (2002) 451e453.
[11] S. Thackray, K. Witte, A.L. Clark, J.G.F. Cleland, Eur. J. Heart Fail. 2 (2000)
209e212.
[12] E.M. Olson, D. Kima, T.W. Smitha, J.D. Marsha, J. Mol. Cell. Cardiol. 19 (1987)
95e104.
[13] L. Mosti, G. Menozzi, P. Schenone, P. Dorigo, R.M. Gaion, F. Benetollo,
G. Bombieri, Eur. J. Med. Chem. 24 (1989) 517e529.
[14] P. Dorigo, R.M. Gaion, P. Bellucco, L. Mosti, P.A. Borea, I. Maragno, Br. J.
Pharmacol. 104 (1991) 867e872.
4.2.1. Cell cultures
The H9C2 cell line (CRL 1446. American Type Culture Collection)
was derived from embryonic BD1X rat heart. The cells weregrown at
subconfluent density in growth medium (GM) Dulbecco’s Modified
Eagle’s Medium (DMEM-F12) containing: 4 mM glutamine, 18 mM
NaHCO3, 25 mM glucose, 1 mM sodium pyruvate, 100 units mLꢀ1
penicillin, 0.1 ng mLꢀ1 streptomycin, supplemented with 10% heat-
inactivated fetal calf serum (FBS, Euroclone). The differentiation was
induced changing the growth medium with differentiative medium
(DM) containing the same elements as growth medium except for
the amount of FCS that was 1% and the adding of 10 nM retinoic acid
to be added every day renewing the DM.
[15] V. Cody, A. Wojtszak, F.B. Davis, P.J. Davis, S.D. Blas, J. Med. Chem. 38 (1995)
1990e1997.
[16] P. Fossa, R. Boggia, E. Lo Presti, L. Mosti, P. Dorigo, M. Floreani, Il Farmaco 52
(1997) 523e530.
[17] M. Floreani, P. Fossa, S. Gessi, L. Mosti, P.A. Borea, P. Dorigo, Naunyn-
Schmiedeberg’s Arch. Pharmacol. 367 (2003) 109e118.
[18] A. Krauze, R. Vitolina, V. Garaliene, L. Sile, V. Klusa, G. Duburs, Eur. J. Med.
Chem. 40 (2005) 1163e1167.
[19] M. De Candia, P. Fossa, S. Cellamare, L. Mosti, A. Carotti, C. Altomare, Eur. J.
Pharm. Sci. 26 (2005) 78e86.
[20] G. Scapin, S.B. Patel, C. Chung, J.P. Varnerin, S.D. Edmondson, A. Mastracchio,
E.R. Parmee, S.B. Singh, J.W. Becker, L.H.T. Van der Ploeg, M.R. Tota,
Biochemistry 43 (2004) 6092e6100.
[21] P.E. Thompson, V. Manganiello, E. Degerman, Curr. Top. Med. Chem. 2 (2007)
421e436.
[22] P. Fossa, F. Giordanetto, G. Menozzi, L. Mosti, Quant. Struct.eAct. Relat. 21
(2002) 267e275.
[23] P. Cage, M. Furber, E. Kinchin, C. Luckhurst, A. Rigby, AstraZeneca R&D, GB,
WO2005/073192, 11.08.2005.
[24] C. Mènard, S. Pupier, D. Mornet, M. Kitzmann, J. Nargeot, P. Lory, J. Biol. Chem.
274 (1999) 29063e29070.
4.2.2. Calcium imaging
At the time of experiments the medium was removed, and cells
were washed twice with Normal External Solution (NES) contain-
ing (in mM): 125 NaCl, 5 KCl, 1 MgSO4, 1 KH2PO4, 5.5 glucose, 1
CaCl2, 20 HEPES, pH ¼ 7.4. Next, the cells were incubated for 40 min
with 5 mM Fura 2-AM. At the end of loading, the cells were washed
with NES and maintained for 15 min at 37 ꢂC to allow the final
hydrolysis of the dye. The Fura trapped in the cell cytoplasm
properly excited at 340 nm and 380 nm emitted its fluorescence at
510 nm that was examined by epifluorescence microscope equip-
ped with monochromator and xenon lamp [29]. In an experimental
set the undifferentiated or differentiated cells were stimulated
directly with the new molecules at a concentration of 10 mM. In
another experimental set the undifferentiated or differentiated
cells were stimulated with 60 mM KCl, a depolarizing salt, in the
[25] H.R. Petty, Microsc. Res. Tech. 8 (2007) 687e709.
[26] T. Pietrangelo, B. Fioretti, R. Mancinelli, L. Catacuzzeno, F. Franciolini, G. Fanò,
S. Fulle, J. Physiol. 572 (2006) 721e733.
presence of 10 mM of the each compound.
[27] H. Hayashi, H. Miyata, J. Pharmacol. Toxicol. Methods 31 (1994) 1e10.
[28] M.V. Berridge, P.M. Herst, A.S. Tan, Biotechnol. Annu. Rev. 11 (Suppl.) (2005)
127e152.
[29] C. Maccallini, T. Pietrangelo, R. Mancinelli, R. Amoroso, G. Bettoni, S. Fulle,
Nitric Oxide 18 (2008) 168e175.
4.2.3. Cellular vitality
The undifferentiated and differentiated cells (9e14 days of
differentiation) were plated in 96-well plates and when they were